Login required to started new threads

Login required to post replies

Beke's test...
Quote | Reply
if jhc or someone else can translate in layman's terms...I aka 'the smartest guy in the world or just on slowtwitch because I said something noone understood' is having a hard time parsing all that...because the lingo ain't mine...


Haematologica. 2001 Feb;86(2):128-37.

Detection of recombinant human erythropoietin abuse in athletes utilizing markers of altered erythropoiesis.

Parisotto R, Wu M, Ashenden MJ, Emslie KR, Gore CJ, Howe C, Kazlauskas R, Sharpe K, Trout GJ, Xie M.

Department of Physiology, Australian Institute of Sport, P.O. Box 176, Belconnen ACT 2616, Australia. robin.parisotto@ausport.gov.au

BACKGROUND AND OBJECTIVES: The detection of recombinant human erythropoietin (r-HuEPO) abuse by athletes remains problematic. The main aim of this study was to demonstrate that the five indirect markers of altered erythropoiesis identified in our earlier work were reliable evidence of current or recently discontinued r-HuEPO use. A subsidiary aim was to refine weightings of the five markers in the initial model using a much larger data set than in the pilot study. A final aim was to verify that the hematologic response to r-HuEPO did not differ between Caucasian and Asiatic subjects. DESIGN AND METHODS: Recreational athletes resident in Sydney, Australia (Sydney, n = 49; 16 women, 33 men) or Beijing, China (Beijing, n=24; 12 women, 12 men) were randomly assigned to r-HuEPO or placebo groups prior to a 25 day administration phase. Injections of r-HuEPO (or saline) were administered double-blind at a dose of 50 IU/kg three times per week, with oral iron (105 mg) or placebo supplements taken daily by all subjects. Blood profiles were monitored during and for 4 weeks after drug administration for hematocrit (Hct), reticulocyte hematocrit (RetHct), percent macrocytes (%Macro), serum erythropoietin (EPO) and soluble transferrin receptor (sTfr), since we had previously shown that these five variables were indicative of r-HuEPO use. RESULTS. The changes in Hct, RetHct, %Macro, EPO and sTfr in the Sydney trial were qualitatively very similar to the changes noted in our previous administration trial involving recreational athletes of similar genetic origin. Statistical models developed from Fisher's discriminant analysis were able to categorize the user and placebo groups correctly. The same hematologic response was demonstrated in Beijing athletes also administered r-HuEPO. INTERPRETATION AND CONCLUSIONS: This paper confirms that r-HuEPO administration causes a predictable and reproducible hematologic response. These markers are disturbed both during and for several weeks following r-HuEPO administration. This work establishes an indirect blood test which offers a useful means of detecting and deterring r-HuEPO abuse. Ethnicity did not influence the markers identified as being able to detect athletes who abuse r-HuEPO.

---------------------------------------
this was the original paper. Apparently RB produces the molecules used as markers, naturally, and quite abundantly during effort.
---------------------------------------

A new and improved test is available apparently:

---------------------------------------
New urinary EPO drug testing method using two-dimensional gel electrophoresis


Alamgir Khan, , , Jasmine Grinyer1, Son T. Truong, Ed J. Breen and Nicolle H. Packer

Proteome Systems Ltd., 1/35-41 Waterloo Road, North Ryde NSW 2113 Australia

Received 25 November 2004; revised 15 February 2005; accepted 15 February 2005. Available online 23 May 2005.



Abstract

We present a two-dimensional electrophoresis (2DE) method for the detection of the drug, recombinant erythropoietin (rHuEPO) in urine and its separation from endogenous erythropoietin (HuEPO). This method involves a one-step acetonitrile precipitation of the proteins in urine, addition of an internal standard, two-dimensional gel electrophoresis (2D PAGE), a single Western blot and chemiluminescent immunodetection.

Results

The 2DE method separates HuEPO and rHuEPO isoforms by both iso-electric point and molecular mass. We have identified several urinary proteins with which the monoclonal EPO antibody used in the current test has non-specific binding. The iso-electric points of these cross-reactive proteins overlap with HuEPO and rHuEPO however, they separate distinctly by the 2DE method. Alpha-2-HS-glycoprotein (HSGP) was identified by peptide mass fingerprinting as one of the urinary cross-reacting proteins, and commercially available purified HSGP was chosen to be added into urine samples as an internal standard prior to separation. Software (EpIQ) was specifically developed that applies four separate criteria to the detection of the migration of rHuEPO and HuEPO relative to the internal standard.

Conclusion

The combination of sample preparation, two-dimensional separation, internal standard, standardized blotting procedures and image analysis software enables the 2DE test for rHuEPO in urine to be performed reproducibly and accurately.

Keywords: Doping; Urine; Erythropoietin; 2DE; EPO detection; Image analysis
Quote Reply
Re: Beke's test... [Francois] [ In reply to ]
Quote | Reply
Test 1 - indirect method by looking at predictable changes in blood profiles after taking EPOHematocrit (Hct), reticulocyte hematocrit (RetHct), percent macrocytes (%Macro), serum erythropoietin (EPO) and soluble transferrin receptor (sTfr) all go up (problem is, how to differentiate that from altitude/low oxygen stimulation of endogenous EPO)

Test 2 - directly looking for exogenous EPO in the urine by 2-dimentional gel electrophoresis, where urinary proteins are separated based on molecular mass and pI (isoelectric point ~ charge) like this:



The gel containing all the urinary proteins is probed with an antibody which recognizes EPO. All the forms have the same molecular mass, but different charges (I'm guessing because the endogenous EPO is glycosylated or otherwise modified with (negatively charged) sugars.)



One nice thing about blots compared to ELISA or flow cytometry, is that in addition to specificty of the antobody, you also have size (and in this case pI) to differentiate between specific and non-specific reactivity. You can see nonspecific binding to other proteins (THP, AC, TPI) but they dont migrate to the same positions as EPO and can be exlcluded.

From what the news reports said, it looks like Beke was producing some of the (less glycosyslated?) forms that would be flagged as recombinant EPO by this test.

_______________________________________________
Quote Reply
Re: Beke's test... [Francois] [ In reply to ]
Quote | Reply
BTW, congrats on the smoking bike split and the ballsy finish.

_______________________________________________
Quote Reply
Re: Beke's test... [jhc] [ In reply to ]
Quote | Reply
the bike split was actually a tad faster...my comp said 2h10'. I lost 3'45'' or so when my chain got stuck between the chainring and cranks and got stuck bad...then the volunteers fell sleepy with the heat and at 3 intersections, I just went straight before being told that it was behind...losing an other 1' or so per miss in the process. Unfortunately the map for the course was really small to know the course unless you were a local. after taking the lead, it was much easier as I could just follow the car.
Quote Reply
Re: Beke's test... [jhc] [ In reply to ]
Quote | Reply
Well done, jhc.

"From what the news reports said, it looks like Beke was producing some of the (less glycosyslated?) forms that would be flagged as recombinant EPO by this test".

Just went to his website and read through the "barrage" of arguments. They don't match up very well. He claims he produces more EPO. He claims he has extraordinary high cross-reactiv protein levels. He claims bacterial contamination.

Which one is it??

I would throw all of that out, unless I have seen the tests (as shown by JHC) in person. Those arguments individually can easily be confirmed or excluded with tests available to the labs.

Same is true for the argument of a different glycolsylation pattern. Can easily be thrown out if other proteins are glycosylated normally (the glycosylaton pattern is constant within one individual).

The most appealing argument is that the sample wasn't stored properly. This will affect glycosylation, which is relatively unstable. This again is very easy to assay.




For the general audience, who doesn't really care about the technical stuff (which in my opinion is very sound and peer-reviewed), I found the following interview from 2003 but which still holds true:



We present to you some of the results of our conversation with Dr. Catlin, coupled with our own research into EPO and drug testing in general, to hopefully answer many of the common questions you may have about EPO and drug testing. Far too many anti-drug crusaders (including ourselves), have never really truly understood how the process works.

Question:
1) EPO is a naturally occurring substance in the body. Could an athlete who lives at altitude or has great genetics and thus might have more EPO in their body naturally than a normal person, test positive for EPO?


No. Currently, to be convicted of an EPO offense athletes must test positive for EPO with the urine EPO test. The urine EPO test is not an indirect test that detects unusually high EPO levels. Rather, it is a direct test that detects the actual presence of recombinant EPO (EPO from a source outside the body). Thus, it would be foolish for an athlete to argue that the test was just showing a naturally high level of natural EPO. As Dr. Catlin said to us, with the urine EPO test the testers "see a footprint of the (recombinant EPO) molecule". The World Anti-Doping Report of March 11, 2003, evaluating the urine EPO test concluded, "the urine EPO test is the only existing test to directly evaluate and prove the EPO abuse of athletes"

Question:
2) I've heard a lot about using both a blood and urine test to detect EPO use. Doesn't an athlete have to test positive for EPO on both the blood and urine test to be considered a doper?

No.

Blood testing has received a lot of attention because it is a new concept in the drug testing world. There is a blood test for EPO use, but it is only an indirect test that can be used as a screening measure to save money by determining whether the urine EPO test needs to be conducted. All the blood test does is tell the testers that the athlete has an unusual blood profile that warrants further investigation. The abnormal profile could be caused by the use of EPO, some other blood boosting drug, or just be explained by the athlete being a genetic freak or living at altitude. The testers then perform the urine EPO test to determine whether artificial EPO is the cause of the abnormality.

The blood test does not have to be done in order for the athlete to test positive for EPO.

Question:
3) If the blood test doesn't have to be performed, then why does it exist? Not only do you say it is unnecessary, but it seems quite invasive and expensive to test athletes' blood when a simple urine test could be done.


Believe it or not, the blood EPO test is much cheaper than the urine EPO test. The blood test costs somewhere in the ballpark of $60, whereas the urine test costs approximately $400 per test. The reason for this is that conducting the urine EPO test takes up a lot of the time of the technicians in the lab (sometimes up to two or three days) . Thus, the blood urine test can be used in situations to save money.

If would be very expensive to conduct a urine EPO tests on all athletes at $400 a pop. Thus the blood EPO test can be used to determine which athletes are most likely to be on EPO, and then the urine EPO test can be administered on this smaller sub sample.

For example purposes only, assume there are 100 athletes and only 1 has used EPO. It would cost $40,000 ($400 *100=$40,000) to test all of them for EPO using the urine EPO test. Instead if the blood test can be used on all the athletes to determine which 10 athletes are most likely to be EPO users, then the expensive urine test can be administered on the 10 suspect athletes and a lot of money can be saved. Combining the blood and the urine test only costs $10,000 ($60*100= $6,000 for the blood test on all the athletes and then 10 * $400= $4000 for the urine test on the 10 suspect athletes).Thus in this hypothetical example, using both the blood and urine test together would save approximately $30,000 as the blood and urine test combination costs only $10,000 versus the $40,000 it would cost to do the urine test on all the athletes.

Question:
4) Are you positively sure about the blood EPO test being unnecessary? Didn't Olga Yegerova get her positive EPO test in 2001 thrown out because a blood test wasn't done?


Yes we're sure the blood test isn't necessary and yes she did get the test thrown out because a blood test wasn't done. But let us explain. When the EPO testing was done in Sydney the protocol was to do the blood testing to first screen the samples and then use the urine test to actually test for EPO. It took a while for the scientific community and the sports federations to finally agree that the urine test in itself was the only test necessary for a positive EPO test. In 2001, Yegerova's urine tested positive and the French accepted this as conclusive proof of a positive test, but the IAAF still insisted that a blood test was needed (her blood had not been taken so a blood test could not be done) as well even though in reality the blood test is only used as a screening device and not necessary for a positive test. Ironically, when Yegerova's blood was screened at the World Championships a few weeks later, her blood sample came back as suspicious, but the urine test did not show EPO usage (remember the urine EPO test can often only detect EPO usage in the previous 48 hours).

Question:
5) What about an athlete testing falsely positive? Isn't that a possibility.


It's highly, highly unlikely if proper procedures are carried out. As Dr. Catlin said the urine test is "a very solid test." Only 6 labs in the world as of this past March carried out the EPO test. Dr. Catlin said, "I don't know how other labs do it (the exact procedures they use in carrying out the test), but we don't call a test positive unless we're absolutely sure". In addition, the March 2003 WADA report evaluating the urine EPO test stated "in its normal use, this test, apparently, has never been found to give false positive results". The test when implemented correctly has a safety margin built in to prevent false positives. Plus remember, in addition to the test being very sound, athletes have 2 samples tested (an "A" and a "B" sample) that both must comeback positive before an athlete is determined to have committed a doping offense. At this stage, only Mr. Lagat's "a" sample has come back positive. He will be entitled to have his own representatives present to ensure proper protocols are followed when his "b" sample is tested in the time intensive urine EPO testing process.

Remember the urine EPO test when properly administered is a test for direct proof of recombinant EPO. Often in the press, we read about athletes having drug tests over turned for not being conclusive, but often times this is when indirect evidence (say a high T/E ratio) is used to determine a drug positive.

6) How effective is the urine EPO test? Won't the drug cheats always be figuring out ways to beat the test?

The bad news is that the urine EPO test currently doesn't detect EPO usage very far back in time. Dr. Catlin noted that how effective the test is in detecting past EPO use is highly variable and depends on the dose of EPO the person is taking, their metabolism and other factors. It is common for the urine test to only detect EPO use in the last 48 hours but for some individuals the test can detect EPO use up to 6 or 7 days in the past. Around 100 athletes at the Salt Lake Olympics who had abnormal values from the blood EPO test (indicating that they were possibly doping), passed the urine EPO test. Presumably some or many of these 100 athletes were dopers, but passed the urine EPO test only because currently the urine EPO test can only detect very recent EPO use. As Dr. Catlin said in the March 20, 2002, Washington Post about these 100 suspicious people, "My guess is that we were looking at people who had used, but their urine [sample] turned up negative because EPO goes away very fast."

The good news is researches are slowly making the urine EPO test more effective (so it will go back farther in time looking for EPO usage) and less expensive. The bad news is it costs a lot of money to do the scientific research to make the tests more effective and the anti-doping researchers are vastly underfunded according to Dr. Catlin. Oftentimes in the press the anti-drug crusade is presented as a futile exercise where the cheats will always be ahead of the testers. But according to Dr. Catlin, the main reason the testers are always behind the drug cheats is because a lack of funding.

As he said, "The main frustration I have is that we could be ahead (of the drug cheaters) all the time, but it takes money." Ultimately he is convinced that there is enough money within world sport to combat the drug problem but it is a matter of convincing people in sport to spend the money on drug testing and research. "If they want to solve the problem (of drugs in sport) they will. But I'm not sure it's going to happen."



This should clear up some things without confusing the general audience.



adrialin

(BOMK, racing drug and supplement free since 1985)
Last edited by: adrialin: Aug 9, 05 10:50
Quote Reply
Re: Beke's test... [adrialin] [ In reply to ]
Quote | Reply
Interesting thought about improper handling.

What do you think happened? (from cyclingnews)

Researchers at the Catholic University in Leuven, Belgium (KUL) have cast doubt on the EPO-detecting urine test currently in use by the UCI. The researchers say that the test in potentially unreliable because it traces too many types of protein, leading to the risk of a false positive. After a race it is possible for athlete to excrete some of the proteins detected by the test, causing a positive result without the use of EPO.

_______________________________________________
Quote Reply